HIV-1 infection is epidemic world wide, causing a variety of immune system-failure related phenomena commonly termed acquired immune deficiency syndrome (AIDS). Recent studies of the dynamics of HIV replication in patients under antiviral therapy have reaffirmed the central role of the virus in disease progression, and provide a strong rationale for the development of effective, long term antiviral therapy (Coffin, J. M. Science, (1995) 267:483-489; Ho et al., Nature (1995) 373:123-6; Wei etal., Nature (1995) 373:117-22).
One interesting parameter from these studies is the extremely short life span of an HIV-1 infected CD4+lymphocyte (half life=1-2 days), contrasting data from other studies which gave an estimated lifespan of months to years for uninfected lymphocytes (Bordignon et al., Hum Gene Ther. (1993) 4:513-20). These observations are relevant for intracellular immunization and antiviral gene therapy, because cells resistant to viral infection, or which suppress viral replication, are strongly selected for in vivo.
The molecular receptor for HIV is the surface glycoprotein CD4 found mainly on a subset of T cells, monocytes, macrophage and some brain cells. HIV has a lipid envelope with viral antigens that bind the CD4 receptor, causing fusion of the viral membrane and the target cell membrane, and release of the HIV capsid into the cytosol. HIV causes death of these immune cells, thereby disabling the immune system and eventually causing death of the patient due to complications associated with a disabled immune system. HIV infection also spreads directly from cell to cell, without an intermediate viral stage. During cell-cell transfer of HIV, a large amount of viral glycoprotein is expressed on the surface of an infected cell, which binds CD4 receptors on uninfected cells, causing cellular fusion. This typically produces an abnormal multinucleate syncytial cell in which HIV is replicated and normal cell functions are suppressed.
Pathogenicity of HIV-1 in vivo appears to be directly related to viral expression levels (for a review see Haynes, et al., Science, 271, 324-328 (1996)). Although drugs such as reverse transcriptase (RT) and protease inhibitors are effective over the short term, because of the emergence of resistance and side effects, their long term use remains problematic. For these reasons, several gene therapy approaches to prevent or interfere with viral replication at different stages of the HIV-1 life cycle are of interest. Antisense oligonucleotides, ribozymes, trans-dominant negative mutants of HIV-1 gene products, inducible suicide genes, intracellularly expressed antibodies against viral proteins, and molecular decoys for the Tat-inducible response region (TAR) and Rev responsive elements (RRE) have been used to inhibit HIV-1 replication (for an overview see, e.g., Yu, et al., Gene Therapy, 1, 13-26 (1994)).
More generally, anti-viral therapeutics, including anti-HIV therapeutics, can target, inter alia, viral RNAs (e.g., using ribozymes, or antisense RNA), viral proteins (RNA decoys, transdominant viral proteins, intracellular single chain antibodies, soluble CD4), infectible cells (suicide genes), or the immune system (in vivo immunization). Similar approaches can also be used for making therapeutics against cancer cells, e.g., by targeting oncogene products with ribozymes, transdominant proteins, and ligands such as antibodies which bind proteins encoded by the oncogene. However, all of these therapeutic approaches are hampered by the limitations of the delivery systems currently used to deliver anti-viral or anti-cancer therapeutics, and by the therapeutics themselves.
For instance, with regard to HIV treatment, the extensively used murine retroviral vectors transduce human peripheral blood lymphocytes poorly, and fail to transduce non-dividing cells such as monocytes/macrophages, which are known to be reservoirs or mediators of many viral infections and cancerous conditions. An appealing alternative basis for therapeutic vectors would be to utilize HIV-based delivery systems, which would ensure optimal CD4+cell targeting and intracellular co-localization of HIV target and gene therapeutic effector molecules. In addition, HIV-derived vectors could be packaged by wild type HIV virions of HIV-infected patients in vivo, and thereby be replicated and disseminated to a larger pool of potentially HIV-infectible cells upon infection by HIV. Some of the regulatory elements which could be used in such vectors (e.g. TAR, RRE and packaging signal sequences) would themselves be antagonistic to HIV replication (i.e., they would act as molecular decoys), thereby providing an additional level of HIV inhibition.
The capacity to infect quiescent cells, which is not shared by oncoretroviruses or MoMLV-derived retroviral vectors, also provides the possibility of using HIV-based vectors to target therapeutics for treatment of other viral conditions and of various cancers. HIV-based vectors which stably transfer genes to rarely dividing stem cells and post-mitotic cells in the hematopoietic, nervous, and other body systems are desirable. Such vectors could be used to treat HIV infections, and many other disorders which are mediated by target cells infectable by HIV, or transducible by HIV-based vectors.
HIV cell transformation vectors can be used to transduce non-dividing hematopoietic stem cells (CD34+), e.g., by pseudotyping the vector. These stem cells differentiate into a variety of immune cells, including CD4+cells which are the primary targets for HIV infection. CD34+ cells are a good target for ex vivo gene therapy, because the cells differentiate into many different cell types, and because the cells are capable of re-engraftment into a patient undergoing ex vivo therapy. The vesicular stomatitis virus envelope glycoprotein (VSV-G) has been used to construct VSV-G-pseudotyped HIV vectors which can infect hematopoietic stem cells (Naldini et al. (1996) Science 272:263 and Aldina et al. (1996) J. Virol 70:2581).
Existing vectors and therapeutics have several features which could be improved. One is the narrow specificity of the antiviral molecules, which can have a limited beneficial effect when considered in light of the genetic plasticity of HIV-1. Resistant variants may arise, similar to the situation with more common anti-viral drugs. A second problem is loss of expression of anti-viral genes, which can occur against antiviral proteins because of immune responses against foreign therapeutic proteins. Loss of expression can also occur with polymeric TAR and RRE molecules by deletion through recombination. A third problem is that expression of protective gene is optionally regulated to occur only when needed, i.e., in infected cells, in order to minimize unintended side effects.
Accordingly, there is a need for improved HIV-based vectors for delivering existing anti-viral genes to cells in vitro, ex vivo and in vivo, and for improved therapeutics against viruses which infect cells transduced by HIV-based vectors (including HIV), and against cancer and other disorders which occur in, or are mediated by, cells which can be transduced by HIV-based vectors. This invention fulfills these and other needs.